Oligodendrocytes are central nervous system cells present in vertebrates. They produce a laminated, lipid-rich myelin sheath that wraps the neuronal axons and creates defined segments of electrical insulation to maximize the speed of action potential conduction. Myelin is also important for axonal integrity and survival, and it has been shown that even small changes affecting oligodendrocyte metabolism can lead to neurodegeneration. Kassmann, C. M. et al., Nature Genet. 39:969-976 (2007). The myelination process is particularly important in humans, as human brains have a higher content of myelinated neurons (white matter) and myelination continues after birth and throughout life. Bartzokis, G., Adolescent Psychiat. 29:55-96 (2005). This suggests that oligodendrocytes are not merely providing inert insulation, but rather that myelination is a dynamic process, affecting cognitive function and even behavior.
Multiple sclerosis (MS), adrenoleukodystrophy, vanishing white matter disease, Pelizaeus-Merzbacher disease, and leukodystrophies are examples of demyelinating or dysmyelinating disorders. In addition, a critical role for oligodendrocytes is emerging in many other neurological disorders and neurodegenerative conditions, including amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, and schizophrenia. Bernstein, H. G. et al., Schizophr. Res. 161:4-18 (2015); Behrendt, G. et al., Glia 61:273-286 (2013); Kang, J. et al., Ann. Vasc. Surg. 27:487-496 (2013); Fennema-Notestine, C. et al., Neurology 63:989-995 (2004). Human oligodendrocyte progenitor cells (OPCs) can be used to develop in vitro myelination assays, to screen for myelinating compounds, and ultimately could become a source for autologous cell replacement therapies. In particular, the generation of patient-specific cells from induced pluripotent stem cells (iPSCs) for autologous cell therapy has recently emerged as a promising concept. Wang, S. et al., Cell Stem Cell 12:252-264 (2013); Goldman, S. A. et al., Science 338:491-495 (2012).
Studies of human oligodendrocyte biology and human myelination have been hampered by the limited access to primary cells from biopsies or autopsies. Pluripotent stem cells (PSCs) have been used within the last two decades as an alternative, useful source from which any desired cell type can be generated. This has been achieved by recapitulating in vitro the fundamental steps of embryonic development. Irion, S. et al., Cold Spring Harb. Sym. 73:101-110 (2008). Notably, most of the critical pathways of lineage commitment are highly conserved between mice and humans and therefore, insights gained from mouse developmental biology have been successfully applied to produce numerous cell types, including oligodendrocytes, from human PSCs (hPSCs). Murry, C. E. et al., Cell 132:661-680 (2008).
In mice, oligodendrocyte precursors arise within the motor neuron progenitor (pMN) domain. During mouse embryonic development, retinoic acid (RA) and sonic hedgehog (SHE) pathways are critical in defining the pMN domain. This finding has been exploited in in vitro cultures to convert neuroepithelial cells into progenitors of the pMN domain, expressing the transcription factor OLIG2. Wichterle, H. et al., Cell 110:385-397 (2002). RA at a concentration of 10 μM has been used in most methods to differentiate human embryonic stem cells (hESCs) to oligodendrocytes. Izrael, M. et al., Mol. Cell. Neurosci. 34:310-323 (2007); Nistor, G. I. et al., Glia 49:385-396 (2005). The Zhang laboratory demonstrated that SHH is necessary for the induction of OLIG2+ NKX2.2+ progenitors, and for their transition to oligodendrocyte progenitor cells (OPCs) expressing SOX10, PDGFRα and ultimately O4. Hu, B. Y. et al., Development 136:1443-1452 (2009a). The appearance of OPCs in vitro is significantly delayed in the human model compared to the mouse model; there is a protracted period of 10 weeks between the peak of OLIG2+ progenitors and the peak of OPCs in the human model. Hu, B. Y. et al., Nat. Protoc. 4:1614-1622 (2009b).
Several groups have devised protocols to differentiate PSCs to OPCs. Numasawa-Kuroiwa, Y. et al.; Stem Cell Rep. 2:648-661 (2014); Stacpoole, S. R. et al., Stem Cell Rep. 1:437-450 (2013); Wang et al., (2013); Liu, Y. et al., Nat. Protoc. 6:640-655 (2011); Sim, F. J. et al., Nat. Biotechnol. 29:934-941 (2011). However, these differentiation protocols are lengthy and inefficient; their practical application is limited by required culture times of over 120 days to obtain OPCs expressing the O4 antigen. Furthermore, the reported efficiencies of O4+ cells obtained range from 4% to 47%. In addition, many differentiation protocols have been optimized using only one or two hESC lines, and their reproducibility with iPSC lines is controversial. Alsanie, W. et al., Stem Cells Devel. 22:2459-2476 (2013).
Therefore, an improved oligodendrocyte differentiation protocol that generates large numbers of purified OPCs in a relatively short time is highly desirable. Moreover, this protocol should be reproducible among different PSC lines and should be highly efficient.